Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

This article describes a methodology for the isolation, characterization, and quantification of human plasma-derived extracellular vesicles (EV) and presents a workflow for label-free analysis of the EV proteome using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research due to their role in regulating intercellular communication, potentially serving as valuable biomarkers for cardiovascular disease. However, the EV proteome and its potential as a biomarker in cardiovascular diagnostics remain poorly understood. This protocol presents a standardized method for the isolation and quantification of plasma-derived EV and the analysis of their protein cargo using plasma samples from patients presenting to the Chest Pain Unit of a large university hospital. Following routine phlebotomy, EV are isolated from plasma by differential ultracentrifugation. The enrichment of specific EV marker proteins in EV isolates is visualized by immunoblotting, and average size distribution and plasma EV concentrations are quantified by nanoparticle tracking analysis. Finally, ultra-performance liquid chromatography-tandem mass spectrometry is employed for label-free analysis of the EV proteome. This protocol thus provides a comprehensive approach to study and use plasma-derived EV as potential carriers of critical biological information as well as to explore their potential as novel biomarkers.

Extracellular vesicles (EV), by nomenclature not uniformly defined, are nanoparticles surrounded by a lipid bilayer and released by various cell types, lacking the ability to replicate¹. This diverse group includes exosomes, a subpopulation of EV of endosomal origin, typically ranging from approximately 40 nm to 160 nm in diameter². Detectable in numerous body fluids³, EVs facilitate intercellular communication by transferring various active biomolecules such as proteins, mRNA, microRNA, and lipids. Thus, EV provide information about their cell of origin through cell-specific surface markers and b....

For this protocol, an exemplary patient cohort was recruited comprising three healthy control patients with no signs of apparent or underlying cardiovascular disease and two patients with non-ST elevated myocardial infarction (NSTEMI). Prior approval was granted by the Institutional Review Board of the Medical Faculty of the University of Heidelberg (IRB approval #S-351/2015), and written consent was obtained from all patients prior to recruitment. Patients were recruited from the Chest Pain Unit of the Department of Car.......

EV were isolated from plasma samples (n = 3) of patients without overt cardiovascular disease, as well as from patients with non-ST elevation myocardial infarction (NSTEMI; n = 2), using the established differential ultracentrifugation protocol. Adequate separation of plasma EV was confirmed by immunoblotting of EV-enriched proteins TSG-101, annexin 5 (Anx5), and CD9 in EV isolates compared to EV-depleted plasma from the same patients (Figure 1A). Transferrin, serving as a negative control, .......

This protocol provides a real-world, step-by-step, ready-to-use methodology for the separation and characterization of plasma EV, as well as an introduction to an unlabeled proteome analysis suitable for integration into routine clinical practice. A detailed description and strict adherence to a unified methodology for EV isolation from plasma samples are important to ensure reproducibility of obtained results. Current literature indicates that pre-analytical conditions can significantly impact subsequent measurements an.......

EG received honoraria for lecturers from Roche Diagnostics, BRAHMS Thermo Scientific, Bayer Vital GmbH, AstraZeneca, Lilly Deutschland, Boehringer Ingelheim; he received institutional research grants from Roche Diagnostics and Daiichi Sankyo, and serves as a consultant for Roche Diagnostics, BRAHMS Thermo Scientific, Astra Zeneca, Novartis and Boehringer Ingelheim, outside the submitted work. JBK received project-related funding from the German Centre for Cardiovascular Research (DZHK) and Roche Diagnostics. The remaining authors declare no conflict of interest related to the submitted work.

The authors thank Heidi Deigentasch, Amelie Werner, and Elisabeth Mertz for their organizational support during this project.